Immune responses protect a human body from pathogens, and the immune system is composed of many immune-related cells and cytokines. Leukocytes, especially lymphocytes, play an important role in the immune system. Representative examples of cells constituting the lymphocytes include innate immune system cells and acquired immune system cells. Natural killer cells (NK cells) are one of the representative innate immune cells, and known as cells that can kill cancer in a non-specific manner, recognize and kill viruses, bacteria, and the like, and kill pathogens with enzymes such as perforins and granzymes or by Fas-FasL interaction. In the case of cancer patients, a decrease in the ability of such NK cells to kill cancer cells is reported to be deeply associated with the onset of diseases such as lung cancer (Carrega P, et al., Cancer, 2008: 112: 863-875), liver cancer (Jinushi M, et al., J Hepatol., 2005: 43; 1013-1020), breast cancer (Bauernhofer T, et al., Eur J Immunol., 2003: 33: 119-124), uterine cancer (Mocchegiani E., et al., Br j Cancer., 1999: 79: 244-250), blood cancer (Tajima F., et al, Lekemia 1996: 10: 478-482), and the like. Therefore, an increase in the ability and activity of the natural killer cells in regards to killing the cancer cells in cancer patients is essential for cancer treatment. Attempts to treat solid cancer or blood cancer are being currently conducted using such an ability of the NK cells to kill the cancer cells.
To obtain an effect of killing cancer cells, a large quantity of NK cells are needed, but it is difficult to ensure obtaining a large amount of blood from cancer patients, and the NK cells in the blood merely amount to a proportion of approximately 5 to 20%. Thus, since it is difficult to use the NK cells as an immunotherapeutic agent, it is important to extend and proliferate the NK cells effectively. A conventional method of expanding the NK cells typically includes separating or inducing NK cells from bone marrow or mononuclear cells in the blood using an apparatus such as a magnetic activated cell sorter (MACS), cliniMACS, or a fluorescence activated cell sorter (FACS). In such methods, the following operations proceed as follows: 1) separating NK cells from mononuclear cells at early stages and expansion-culturing the NK cells using cytokines; 2) removing T cells coexisting with the mononuclear cells and expansion-culturing the NK cells using cytokines; and 3) inducing the NK cells from stem cells present in the bone marrow. In addition, as the method of isolating NK cells from peripheral blood mononuclear cells (PBMCs) using feeder cells, a method using a RPMI8866 cell line that is derived from B cell leukemia by the Torelli research team from the Republic of Italy, and a method using a HFWT cell line that is derived from a Wilms tumor cell by the Ishikawa research team from Japan have been reported.
However, methods for expanding NK cells reported in the related art are encumbered by the requirement for sophisticated equipment to select cells pre-expansion, the use of genetically engineered feeder cells, and a cocktail of costly cytokines in high concentrations, and thus may be prohibitively expensive for general use, are more difficult to implement and limit accessibility of this approach to the patient population.